影响糖尿病大鼠骨折愈合相关因素及BMP-2、IGF-I干预治疗的实验研究
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摘要
目的观察和检测糖尿病大鼠骨折模型骨折愈合,骨密度变化及BMP-2、IGF-Ⅰ、Ⅰ型胶原、骨钙素、碱性磷酸酶在骨折局部和血清中的表达情况。研究骨折局部使用BMP-2、IGF-Ⅰ促进糖尿病大鼠骨折愈合的效果。探讨影响糖尿病大鼠骨折愈合的相关因素,及BMP-2、IGF-Ⅰ促进糖尿病大鼠骨折愈合的作用。
方法本研究分为两个部分:
第一部分,影响糖尿病大鼠骨折愈合相关因素研究。实际用于检测的大鼠为雄性3月SD大鼠60只,体重200—250g,随机平均分为2组,每组30只,A组为实验组(糖尿病大鼠骨折组);B组为对照组(正常大鼠骨折组)。中途死亡和造模失败的大鼠再行造模补充,保证每次每组用于检测的大鼠为5只。A组用链脲佐菌霉素(STZ)破坏胰岛B细胞诱导为糖尿病大鼠,B组进行对照。无菌操作下用剪刀剪断两组大鼠左胫骨上段,然后复位外固定。分别于术后第1、2、3、4、6、8周拍大鼠左下肢X线片,腰椎、髋关节、骨折端局部骨和骨痂进行骨密度检测,观察骨折愈合情况;取骨折断端组织行HE染色光镜观察,抽血测血糖、钙、磷、碱性磷酸酶,ELISA法检测血清BMP-2、IGF-Ⅰ,放射免疫检测血清骨钙素,免疫组化检测断端骨痂组织BMP-2、IGF-Ⅰ、骨钙素(OC)表达,实时荧光定量RT—PCR检测断端骨痂BMP-2、IGF-Ⅰ和Ⅰ型胶原蛋白表达含量;对以上指标,A、B组进行比较。
第二部分,BMP-2、IGF-Ⅰ在糖尿病大鼠骨折愈合过程中的干预治疗作用研究。实际用于检测的大鼠为30只,条件同前,同上处理为糖尿病大鼠骨折模型。随机平均分为2组,每组15只,A′组为糖尿病大鼠骨折后用IGF-Ⅰ复合纤维蛋白胶治疗组;B′组为糖尿病大鼠左胫骨骨折后用BMP-2复合纤维蛋白胶治疗组。中途死亡和造模失败的大鼠再行造模补充,保证每次每组用于检测的大鼠为5只。分别于术后第2、4、6周进行检测。拍大鼠左下肢X线片,局部骨和骨痂骨密度检测,观察骨折愈合情况;取骨折断端组织行HE染色光镜观察,抽血测血糖、碱性磷酸酶,放射免疫检测血清骨钙素,免疫组化检测局部骨痂BMP-2、IGF-Ⅰ、骨钙素(OC)含量,实时荧光定量RT—PCR检测局部骨痂Ⅰ型胶原蛋白表达含量。对以上指标,A′、B′组同第一部分A组进行比较,A′、B′组再相互比较。
运用SPSS11.5进行数据录入和统计处理,采用成组t检验和单因素方差分析方法进行分析。
结果实验结果显示:
第一部分与正常大鼠骨折组(B组)比较,糖尿病大鼠骨折组(A组),在术后1、2、3、4、6、8周:1)血糖明显升高,均大于13.8mmol/L;2)血钙、磷含量下降,第6周差距最大;3)血清BMP-2、IGF-Ⅰ、碱性磷酸酶、骨钙素分泌明显减少,高峰均推迟一周;4)X线片上骨痂生成量均减少;5)腰椎、髋关节、骨折端骨和骨痂组织骨密度下降,第4周后下降明显,与B组比较第八周差距最大;6)光镜下HE染色观察骨形成滞后;7)免疫组化示:断端骨痂BMP-2、IGF-Ⅰ、骨钙素表达下降;8)实时荧光定量RT—PCR检测:断端骨痂组织BMP-2、IGF-Ⅰ和Ⅰ型胶原蛋白1—3周表达均下降,组间差异有显著性,第2周时差异最大,BMP-2、IGF-Ⅰ第4周无明显差异,高峰均推迟一周,Ⅰ型胶原蛋白第4、6周表达下降,仍有差异,第8周无明显差异。
第二部分与糖尿病大鼠骨折未治疗组(A组)相比,糖尿病大鼠骨折后用IGF-Ⅰ复合纤维蛋白胶治疗组(A′组)和糖尿病大鼠骨折后用BMP-2复合纤维蛋白胶治疗组(B′组):1)血清碱性磷酸酶、骨钙素表达明显升高,在第二周表达最高,第六周无明显差异;2)X线片上骨痂生成量均增多,局部骨和骨痂组织密度略升高,差异无统计学意义;3)实时荧光定量RT—PCR检测局部骨痂组织Ⅰ型胶原蛋白表达升高,第二周时差异最大,第六周无明显差异。和B′组比较,A′组实时荧光定量RT—PCR检测局部骨痂组织Ⅰ型胶原蛋白表达略升高,第二周时差异最大,第六周无明显差异。
结论1)糖尿病大鼠骨折后骨折愈合差,出现愈合延迟或不愈合;2)糖尿病大鼠骨折愈合过程中早期血清及局部骨痂组织中BMP-2、IGF-Ⅰ表达量下降;3)糖尿病大鼠骨折愈合过程中局部骨和骨痂组织中成骨细胞数量和功能下降,Ⅰ型胶原蛋白表达减少;4)糖尿病大鼠钙、磷丢失明显,骨折愈合过程中局部骨和骨痂组织骨密度下降,骨痂矿化障碍;5)糖尿病大鼠骨折愈合过程中早期血清及局部骨痂组织中BMP-2、IGF-Ⅰ下降可以引起成骨细胞数量和功能下降,Ⅰ型胶原蛋白表达减少,后期骨痂矿化障碍,是导致糖尿病大鼠骨折愈合差的重要因素;6)在相同条件和浓度下用IGF-Ⅰ复合纤维蛋白胶和BMP-2复合纤维蛋白胶作用于糖尿病大鼠局部骨折端,均可明显改善骨折愈合情况;7)在相同条件和浓度下,IGF-Ⅰ复合纤维蛋白胶比BMP-2复合纤维蛋白胶作用于糖尿病大鼠骨折愈合效用更为明显;8)局部使用纤维蛋白胶复合BMP-2或IGF-Ⅰ治疗糖尿病大鼠骨折,不能有效提高骨折端局部骨和骨痂组织的骨密度。
Objective:
To investigate the variations of bone mineral density and the expression of BMP-2, IGF-Ⅰ,typeⅠcollagen protein,osteocalcin and alkaline phosphatase in diabete rats in the fracture region and serum during the fracture healing,and to evaluate the effects on promoting fracture healing in diabete rats by using BMP-2,IGF-Ⅰin the fracture region.Then to discuss the relevant factors affected fracture healing and the roles of BMP-2,IGF-Ⅰin promoting fracture healing in diabete rats.
Methods:
The experiment was divided into two parts.
The first part was to investigate the relevant factors affected the fracture healing in diabete rats.60SD rats(male,3 months old,and 250-300g weight) were divided into two groups randomly,30 in each.Group A was experimental group(the diabete rats with fracture),groupB was control group(the rats with fracture,but without diabete). More rats were taken to guarantee that 5 rats used to be examined in each group in each time point for the rats died and the failure model.
Streptozotocin was injected into enterocoelia to destory the B cells of islet of Langerhans in group A to induce diabetes models.Group B was given as the control group.The left tibias of all the models were cutten by shears,and then treated with reduction and external fixation.The X-ray of left lower extremity and the bone mineral density of lumbar spine,hip,local bone and callus were taken in 1st,2nd,3rd, 4th,6th,8th week after the surgery to investigate the healing of fracture,the tissue of fracture region was obtained for HE staining.Draw off blood from arteria cruralis and screened the concentration of glucose,calcium,phosphorus,and alkaline phosphatase.BMP-2,IGF-Ⅰin serum was examined by ELISA assay. Radioimmunoassay for OC in serum,immunohistochemical method for BMP-2, IGF-Ⅰ,and OC in local callus,and real-time quantitative RT—PCR for the expression of BMP-2,IGF-Ⅰand typeⅠcollagen protein in local callus were used.And contrasted the difference between the two groups.
The second part of the study was to investigate the treatment effects of BMP-2,IGF-Ⅰin the process of fracture healing in diabetes rats.30 rats were divided into two groups randomly as same as part one(A'and B'),15 in each.Group A' was the group of fracture treating by IGF-Ⅰcomplex fibrin glue in diabetes rats,and group B'was by BMP-2 composite fibrin glue.More rats were taken to guarantee that 5 rats used to be examined in each group in each time point for the rats died and the failure model.The X-ray of left lower extremity and the bone mineral density of lumbar spine,hip,local bone and callus were taken in 2nd,4th,6th,week after the surgery to investigate the healing of fracture.The tissue of fracture region was obtained for HE staining.Draw off blood from arteria cruralis and screened the concentration of glucose,calcium, phosphorus,and alkaline phosphatase.Radioimmunoassay for OC in serum, immunohistochemical method for BMP-2,IGF-Ⅰ,and OC callus in local,and real-time quantitative RT—PCR for the expression of typeⅠcollagen protein in local callus were used.And contrast the difference among A',B'and A.
SPSS11.5 statitical package was used to deal with all data.Calculate a p-Value for Student's t-Test and for one-factor analysis of variance.
Results:
In part one,comparation of Group A and B,there were significantly higher increased of blood glucose in the 1st,2nd,3rd,4th,6th,8th week in group A,all over 13.8mmol/L.The content of calcium,phosphoru decreased in group A,and the significantly difference appeared in sixth week.In Serum,secretion of BMP-2,IGF-Ⅰ, alkaline phosphatase,and osteocalcin were significantly decreased and the peak delayed one week later in group A.The callus formation was poor in each time point in group A.Bone mineral density of hip joints,lumbar spine,local bone,and bone callus organization suggested the slight decreased within four weeks,then significant decresed after four weeks,and the significantly difference appeared in eighth week in group A.HE staining under a light microscope suggested the hysteresis of bone formation.Immunohistochemical staining showed the decrease of the expression of BMP-2,IGF-Ⅰ,osteocalcin in group A.Real-time quantitative RT—PCR suggested the expression of BMP-2,IGF-Ⅰand typeⅠcollagen protein in local bone callus was decreased in group A,and the significantly difference appeared in 2rd week,but no significantly difference in expression of BMP-2,IGF-Ⅰin 4th week,and the peak was delayed one week in each group.There were no significantly differences ofⅠcollagen protein between the two groups in the eighth week.
Part two,compared with group A.In Serum,the expression of ALP and osteocalcin were increased significantly in group A' and B',and the highest expression of them were appeared in the second week,but there were no significant differences of them in the sixth week.X-ray suggested the callus formation in A' and B' was increased in 2 groups in each time point,and density of local bone and callus were increased slightly. The expression of typeⅠcollagen protein in callus was increased fastest in group A' and B' detected by RT—PCR,and the significant differences appeared in the second week,but no significant differences in sixth week.
Conclusion:
1) The healing of the fracture in diabetes rats was not satisfactory,even the delayed or non-union occurred.2) During the healing after fracture in rats with Diabetes,the expression of BMP-2 and IGF-Ⅰboth in the serum and in fracture region were decreased early.3) During fracture healing rats with diabetes,the quantity and the function of osteoblast was declined,and the expression of typeⅠcollagen protein was deceased in local bone and bone callus organization.4)Calcium and phosphorus of rats with diabetic are loss obviously,and during the process of fracture healing.the bone mineral density is decreased in local bone and bone callus organization which caused mineralization obstacles in callus.5) The early decrease of BMP-2 and IGF-Ⅰboth in the serum and in fracture region lead to decline both the function and the quantity of osteoblast,and cause the reducing expression of typeⅠcollagen protein at the same time,which was one of the important reasons of poor fracture healing in rats with diabetes.6)Under the same conditions and the same concentration,the fracture healing in rats with diabetic can be improved significantly by using the IGF-Ⅰcomplex fibrin glue or BMP-2 composite fibrin glue.7) But IGF-Ⅰcomplex fibrin glue had more remarkable effect than BMP-2 composite fibrin glue on fracture rats with diabetes.8)fibrin glue complex BMP-2 or IGF-Ⅰin local treatment of fractures in diabetic rats can not effectively improve the bone mineral density of callus and local bone on the fracture.
方法本研究分为两个部分:
第一部分,影响糖尿病大鼠骨折愈合相关因素研究。实际用于检测的大鼠为雄性3月SD大鼠60只,体重200—250g,随机平均分为2组,每组30只,A组为实验组(糖尿病大鼠骨折组);B组为对照组(正常大鼠骨折组)。中途死亡和造模失败的大鼠再行造模补充,保证每次每组用于检测的大鼠为5只。A组用链脲佐菌霉素(STZ)破坏胰岛B细胞诱导为糖尿病大鼠,B组进行对照。无菌操作下用剪刀剪断两组大鼠左胫骨上段,然后复位外固定。分别于术后第1、2、3、4、6、8周拍大鼠左下肢X线片,腰椎、髋关节、骨折端局部骨和骨痂进行骨密度检测,观察骨折愈合情况;取骨折断端组织行HE染色光镜观察,抽血测血糖、钙、磷、碱性磷酸酶,ELISA法检测血清BMP-2、IGF-Ⅰ,放射免疫检测血清骨钙素,免疫组化检测断端骨痂组织BMP-2、IGF-Ⅰ、骨钙素(OC)表达,实时荧光定量RT—PCR检测断端骨痂BMP-2、IGF-Ⅰ和Ⅰ型胶原蛋白表达含量;对以上指标,A、B组进行比较。
第二部分,BMP-2、IGF-Ⅰ在糖尿病大鼠骨折愈合过程中的干预治疗作用研究。实际用于检测的大鼠为30只,条件同前,同上处理为糖尿病大鼠骨折模型。随机平均分为2组,每组15只,A′组为糖尿病大鼠骨折后用IGF-Ⅰ复合纤维蛋白胶治疗组;B′组为糖尿病大鼠左胫骨骨折后用BMP-2复合纤维蛋白胶治疗组。中途死亡和造模失败的大鼠再行造模补充,保证每次每组用于检测的大鼠为5只。分别于术后第2、4、6周进行检测。拍大鼠左下肢X线片,局部骨和骨痂骨密度检测,观察骨折愈合情况;取骨折断端组织行HE染色光镜观察,抽血测血糖、碱性磷酸酶,放射免疫检测血清骨钙素,免疫组化检测局部骨痂BMP-2、IGF-Ⅰ、骨钙素(OC)含量,实时荧光定量RT—PCR检测局部骨痂Ⅰ型胶原蛋白表达含量。对以上指标,A′、B′组同第一部分A组进行比较,A′、B′组再相互比较。
运用SPSS11.5进行数据录入和统计处理,采用成组t检验和单因素方差分析方法进行分析。
结果实验结果显示:
第一部分与正常大鼠骨折组(B组)比较,糖尿病大鼠骨折组(A组),在术后1、2、3、4、6、8周:1)血糖明显升高,均大于13.8mmol/L;2)血钙、磷含量下降,第6周差距最大;3)血清BMP-2、IGF-Ⅰ、碱性磷酸酶、骨钙素分泌明显减少,高峰均推迟一周;4)X线片上骨痂生成量均减少;5)腰椎、髋关节、骨折端骨和骨痂组织骨密度下降,第4周后下降明显,与B组比较第八周差距最大;6)光镜下HE染色观察骨形成滞后;7)免疫组化示:断端骨痂BMP-2、IGF-Ⅰ、骨钙素表达下降;8)实时荧光定量RT—PCR检测:断端骨痂组织BMP-2、IGF-Ⅰ和Ⅰ型胶原蛋白1—3周表达均下降,组间差异有显著性,第2周时差异最大,BMP-2、IGF-Ⅰ第4周无明显差异,高峰均推迟一周,Ⅰ型胶原蛋白第4、6周表达下降,仍有差异,第8周无明显差异。
第二部分与糖尿病大鼠骨折未治疗组(A组)相比,糖尿病大鼠骨折后用IGF-Ⅰ复合纤维蛋白胶治疗组(A′组)和糖尿病大鼠骨折后用BMP-2复合纤维蛋白胶治疗组(B′组):1)血清碱性磷酸酶、骨钙素表达明显升高,在第二周表达最高,第六周无明显差异;2)X线片上骨痂生成量均增多,局部骨和骨痂组织密度略升高,差异无统计学意义;3)实时荧光定量RT—PCR检测局部骨痂组织Ⅰ型胶原蛋白表达升高,第二周时差异最大,第六周无明显差异。和B′组比较,A′组实时荧光定量RT—PCR检测局部骨痂组织Ⅰ型胶原蛋白表达略升高,第二周时差异最大,第六周无明显差异。
结论1)糖尿病大鼠骨折后骨折愈合差,出现愈合延迟或不愈合;2)糖尿病大鼠骨折愈合过程中早期血清及局部骨痂组织中BMP-2、IGF-Ⅰ表达量下降;3)糖尿病大鼠骨折愈合过程中局部骨和骨痂组织中成骨细胞数量和功能下降,Ⅰ型胶原蛋白表达减少;4)糖尿病大鼠钙、磷丢失明显,骨折愈合过程中局部骨和骨痂组织骨密度下降,骨痂矿化障碍;5)糖尿病大鼠骨折愈合过程中早期血清及局部骨痂组织中BMP-2、IGF-Ⅰ下降可以引起成骨细胞数量和功能下降,Ⅰ型胶原蛋白表达减少,后期骨痂矿化障碍,是导致糖尿病大鼠骨折愈合差的重要因素;6)在相同条件和浓度下用IGF-Ⅰ复合纤维蛋白胶和BMP-2复合纤维蛋白胶作用于糖尿病大鼠局部骨折端,均可明显改善骨折愈合情况;7)在相同条件和浓度下,IGF-Ⅰ复合纤维蛋白胶比BMP-2复合纤维蛋白胶作用于糖尿病大鼠骨折愈合效用更为明显;8)局部使用纤维蛋白胶复合BMP-2或IGF-Ⅰ治疗糖尿病大鼠骨折,不能有效提高骨折端局部骨和骨痂组织的骨密度。
Objective:
To investigate the variations of bone mineral density and the expression of BMP-2, IGF-Ⅰ,typeⅠcollagen protein,osteocalcin and alkaline phosphatase in diabete rats in the fracture region and serum during the fracture healing,and to evaluate the effects on promoting fracture healing in diabete rats by using BMP-2,IGF-Ⅰin the fracture region.Then to discuss the relevant factors affected fracture healing and the roles of BMP-2,IGF-Ⅰin promoting fracture healing in diabete rats.
Methods:
The experiment was divided into two parts.
The first part was to investigate the relevant factors affected the fracture healing in diabete rats.60SD rats(male,3 months old,and 250-300g weight) were divided into two groups randomly,30 in each.Group A was experimental group(the diabete rats with fracture),groupB was control group(the rats with fracture,but without diabete). More rats were taken to guarantee that 5 rats used to be examined in each group in each time point for the rats died and the failure model.
Streptozotocin was injected into enterocoelia to destory the B cells of islet of Langerhans in group A to induce diabetes models.Group B was given as the control group.The left tibias of all the models were cutten by shears,and then treated with reduction and external fixation.The X-ray of left lower extremity and the bone mineral density of lumbar spine,hip,local bone and callus were taken in 1st,2nd,3rd, 4th,6th,8th week after the surgery to investigate the healing of fracture,the tissue of fracture region was obtained for HE staining.Draw off blood from arteria cruralis and screened the concentration of glucose,calcium,phosphorus,and alkaline phosphatase.BMP-2,IGF-Ⅰin serum was examined by ELISA assay. Radioimmunoassay for OC in serum,immunohistochemical method for BMP-2, IGF-Ⅰ,and OC in local callus,and real-time quantitative RT—PCR for the expression of BMP-2,IGF-Ⅰand typeⅠcollagen protein in local callus were used.And contrasted the difference between the two groups.
The second part of the study was to investigate the treatment effects of BMP-2,IGF-Ⅰin the process of fracture healing in diabetes rats.30 rats were divided into two groups randomly as same as part one(A'and B'),15 in each.Group A' was the group of fracture treating by IGF-Ⅰcomplex fibrin glue in diabetes rats,and group B'was by BMP-2 composite fibrin glue.More rats were taken to guarantee that 5 rats used to be examined in each group in each time point for the rats died and the failure model.The X-ray of left lower extremity and the bone mineral density of lumbar spine,hip,local bone and callus were taken in 2nd,4th,6th,week after the surgery to investigate the healing of fracture.The tissue of fracture region was obtained for HE staining.Draw off blood from arteria cruralis and screened the concentration of glucose,calcium, phosphorus,and alkaline phosphatase.Radioimmunoassay for OC in serum, immunohistochemical method for BMP-2,IGF-Ⅰ,and OC callus in local,and real-time quantitative RT—PCR for the expression of typeⅠcollagen protein in local callus were used.And contrast the difference among A',B'and A.
SPSS11.5 statitical package was used to deal with all data.Calculate a p-Value for Student's t-Test and for one-factor analysis of variance.
Results:
In part one,comparation of Group A and B,there were significantly higher increased of blood glucose in the 1st,2nd,3rd,4th,6th,8th week in group A,all over 13.8mmol/L.The content of calcium,phosphoru decreased in group A,and the significantly difference appeared in sixth week.In Serum,secretion of BMP-2,IGF-Ⅰ, alkaline phosphatase,and osteocalcin were significantly decreased and the peak delayed one week later in group A.The callus formation was poor in each time point in group A.Bone mineral density of hip joints,lumbar spine,local bone,and bone callus organization suggested the slight decreased within four weeks,then significant decresed after four weeks,and the significantly difference appeared in eighth week in group A.HE staining under a light microscope suggested the hysteresis of bone formation.Immunohistochemical staining showed the decrease of the expression of BMP-2,IGF-Ⅰ,osteocalcin in group A.Real-time quantitative RT—PCR suggested the expression of BMP-2,IGF-Ⅰand typeⅠcollagen protein in local bone callus was decreased in group A,and the significantly difference appeared in 2rd week,but no significantly difference in expression of BMP-2,IGF-Ⅰin 4th week,and the peak was delayed one week in each group.There were no significantly differences ofⅠcollagen protein between the two groups in the eighth week.
Part two,compared with group A.In Serum,the expression of ALP and osteocalcin were increased significantly in group A' and B',and the highest expression of them were appeared in the second week,but there were no significant differences of them in the sixth week.X-ray suggested the callus formation in A' and B' was increased in 2 groups in each time point,and density of local bone and callus were increased slightly. The expression of typeⅠcollagen protein in callus was increased fastest in group A' and B' detected by RT—PCR,and the significant differences appeared in the second week,but no significant differences in sixth week.
Conclusion:
1) The healing of the fracture in diabetes rats was not satisfactory,even the delayed or non-union occurred.2) During the healing after fracture in rats with Diabetes,the expression of BMP-2 and IGF-Ⅰboth in the serum and in fracture region were decreased early.3) During fracture healing rats with diabetes,the quantity and the function of osteoblast was declined,and the expression of typeⅠcollagen protein was deceased in local bone and bone callus organization.4)Calcium and phosphorus of rats with diabetic are loss obviously,and during the process of fracture healing.the bone mineral density is decreased in local bone and bone callus organization which caused mineralization obstacles in callus.5) The early decrease of BMP-2 and IGF-Ⅰboth in the serum and in fracture region lead to decline both the function and the quantity of osteoblast,and cause the reducing expression of typeⅠcollagen protein at the same time,which was one of the important reasons of poor fracture healing in rats with diabetes.6)Under the same conditions and the same concentration,the fracture healing in rats with diabetic can be improved significantly by using the IGF-Ⅰcomplex fibrin glue or BMP-2 composite fibrin glue.7) But IGF-Ⅰcomplex fibrin glue had more remarkable effect than BMP-2 composite fibrin glue on fracture rats with diabetes.8)fibrin glue complex BMP-2 or IGF-Ⅰin local treatment of fractures in diabetic rats can not effectively improve the bone mineral density of callus and local bone on the fracture.
引文
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